Brucella abortus is a highly infectious intracellular pathogen of humans that is classified as a Category B Bioterrorism agent. Virulence of this organism depends on its ability to survive and replicate within phagosomes of host monocytes. The experiments outlined in this proposal will characterize the maturation of the B. abortus phagosome that becomes acidified but does not fuse with lysosomes. Characterizing how B. abortus phagosomes interact with endosomes, phagosomes and lysosomes will give considerable insight into the host-pathogen relationship. Based on preliminary data and published reports, it is hypothesized that Brucella abortus establishes an intracellular niche within monocytes by interfering with host trafficking machinery to form a modified phagosome that does not fuse with lysosomes. The first set of experiments will use immunofluorescence microscopy and Western blot analysis on purified phagosomes containing live B. abortus to characterize these modified phagosomes. Phagosome maturation is a highly regulated process and key regulators of this pathway are the Rab GTPases Rab5 and Rab7. Altering the activity of these regulators contributes to the survival of other intracellular pathogens by preventing phagosome maturation and fusion with lysosomes. To determine if such a phenomenon is associated with the survival of intracellular B. abortus, phagosome maturation and bacterial survival will be examined in monocytes expressing the dominant-negative and constitutively active forms of Rab5 and Rab7. The goal of these studies is to identify which events in phagosome maturation B. abortus alters for intracellular survival and if Rab5 and Rab7 contribute to or antagonize the development of Brucella's modified phagosome. Completion of these studies will greatly increase our understanding of Brucella pathogenesis that will in turn aid the development a human vaccine and treatments for human brucellosis.